1. Field of the Invention
This invention relates generally to humanized anti-CD11a antibodies.
2. Description of Related Art
Lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is involved in leukocyte adhesion during cellular interactions essential for immunologic responses and inflammation (Larson et al., Immunol. Rev. 114:181-217 (1990)). LFA-1 is a member of the xcex22 integrin family and consists of a unique xcex1 subunit, CD11a, and a xcex2 subunit, CD18, common to other xcex22 integrin receptors Mac-1 and p150,95. The ligands of LFA-1 include intercellular adhesion molecule-1, ICAM-1, expressed on leukocytes, endothelium, and dermal fibroblasts (Dustin et al., J. Immunol. 137:245-254 (1986)), ICAM-2 expressed on resting endothelium and lymphocytes (de Fougerolles et al., J. Exp. Med. 174:253-267 (1991)), and ICAM-3 expressed on monocytes and resting lymphocytes (de Fougerolles et al., J. Exp. Med. 179:619-629 (1994)).
Monoclonal antibodies (MAbs) against LFA-1 an d the ICAMs have been shown, in vitro, to inhibit several T cell-dependent immune functions including T cell activation (Kuypers et al., Res. Immunol. 140:461(1989)), T cell-dependent B cell proliferation (Fischer et al., J. Immunol. 136:3198-3203 (1986)), target cell lysis (Krensky et al., J. Immunol. 131:611-616 (1983)), and adhesion of T cells to vascular endothelium (Lo et al., J. Immunol. 143:3325-3329 (1989)). In mice, anti-CD11a MAbs induce tolerance to protein antigens (Tanaka et al., Eur. J. Immunol. 25:1555-1558 (1995)) and prolong survival of cardiac (Cavazzana-Calvo et al., Transplantation 59:1576-1582 (1995); Nakakura et al., Transplantation 55:412-417 (1993)), bone marrow (Cavazzana-Calvo et al., Transplantation 59:1576-1582 (1995); van Dijken et al., Transplantation 49:882-886 (1990)), corneal (He et al., Invest. Opthamol. Vis. Sci. 35:3218-3225 (1994)), islet (Nishihara et al., Transplantation Proc. 27:372 (1995)) and thyroid (Talento et al., Transplantation 55:418-422 (1993)) allografts.
In humans, anti-CD11a MAbs prevent graft failure after bone marrow transplantation (Fischer et al., Blood 77:249-256 (1991); Stoppa et al., Transplant Intl. 4:3-7 (1991)) and preliminary clinical studies of renal allografts treated prophylactically with anti-CD11a MAb, in addition to corticosteroids and azathioprine, are promising (Hourmant et al., Transplantation 58:377-380 (1994)). Current therapies against graft rejection include use of OKT3, a murine anti-human CD3 MAb, and cyclosporin A. OKT3 therapy is effective but has several undesirable side effects; its use results in the release of numerous cytokines including tumor necrosis factor-xcex1, interferon-xcex3, interleukin-2, and interleukin-6, resulting in fever, chills and gastrointestinal distress (for a review see Parlevliet et al., Transplant Intl. 5:234-246 (1992); Dantal et al., Curr. Opin. Immunol. 3:740-747 (1991)). Cyclosporin A is effective but also has serious side effects (for a review see Barry, Drugs, 44:554-566 (1992)).
The instant invention provides humanized anti-CD11a antibodies. Preferred antibodies bind to the I-domain of human CD11a (e.g. to xe2x80x9cepitope MHM24xe2x80x9d as herein defined) and/or bind CD11a with an affinity of about 1xc3x97108M or stronger. In preferred embodiments, the antibody has an IC50 (nM) value of no more than about 1 nM for preventing adhesion of Jurkat cells to normal human epidermal keratinocytes expressing ICAM-1. Preferred humanized antibodies are those which have an IC50 (nM) value of no more than about 1 nM in the mixed lymphocyte response (MLR) assay. This IC50 for a humanized antibody in the MLR assay is significantly better than that that for murine MAb 25.3, which has been previously tested in vivo (Fischer et al., Blood 77:249-256 (1991); Stoppa et al., Transplant Intl. 4:3-7 (1991); Hourmant et al., Transplantation 58:377-380 (1994)).
The humanized anti-CD11a antibody may have a heavy chain variable region comprising the amino acid sequence of CDR1 (GYSFTGHWMN; SEQ ID NO:10) and/or CDR2 (MIHPSDSETRYNQKFKD; SEQ ID NO:11) and/or CDR3 (GIYFYGTTYFDY; SEQ ID NO:12) of humanized antibody MHM24 F(ab)-8 in FIG. 1 and/or a light chain variable region comprising the amino acid sequence of CDR1 (RASKTISKYLA; SEQ ID NO:13) and/or CDR2 (SGSTLQS; SEQ ID NO:14) and/or CDR3 (QQHNEYPLT; SEQ ID NO:15) of humanized antibody MHM24 F(ab)-8 in FIG. 1. In other embodiments, the antibody comprises an amino acid sequence variant of one or more of the CDRs of humanized MHM24 antibody F(ab)-8, which variant comprises one or more amino acid insertion(s) within or adjacent to a CDR residue and/or deletion(s) within or adjacent to a CDR residue and/or substitution(s) of CDR residue(s) (with substitution(s) being the preferred type of amino acid alteration for generating such variants). Such variants will normally having a binding affinity for human CD11a which is no more than about 1xc3x9710xe2x88x928M.
In preferred embodiments, the humanized antibody includes a light chain variable region comprising the amino acid sequence of SEQ ID NO:2 and/or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5 of humanized antibody MHM24 F(ab)-8 in FIG. 1 and/or amino acid sequence variants thereof.
As described herein, it has been possible to reengineer a humanized antibody that bound human CD11a antigen, but not significantly to rhesus CD11a antigen, so as to confer an ability to bind to rhesus CD11a (i.e. a xe2x80x9crhesusizedxe2x80x9d antibody). In this embodiment, the antibody which binds rhesus CD11a may, for example, comprise the the CDR2 amino acid sequence in SEQ ID NO:23. The other CDRs may be the same as those for humanized MHM24 antibody F(ab)-8. Thus, the antibody may comprise the amino acid sequence of the xe2x80x9crhesusizedxe2x80x9d heavy chain in SEQ ID NO:24, optionally combined with a light chain comprising the amino acid sequence in SEQ ID NO:2.
Various forms of the antibody are contemplated herein. For example, the anti-CD11a antibody may be a full length antibody (e.g. having a human immunoglobulin constant region) or an antibody fragment (e.g. a F(abxe2x80x2)2). Furthermore, the antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound (such as a cytotoxic agent).
Diagnostic and therapeutic uses for the antibody are contemplated. In one diagnostic application, the invention provides a method for determining the presence of CD11a protein comprising exposing a sample suspected of containing the CD11a protein to the anti-CD11a antibody and determining binding of the antibody to the sample. For this use, the invention provides a kit comprising the antibody and instructions for using the antibody to detect the CD11a protein.
The invention further provides: isolated nucleic acid encoding the antibody; a vector comprising that nucleic acid, optionally operably linked to control sequences recognized by a host cell transformed with the vector; a host cell comprising that vector; a process for producing the antibody comprising culturing the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture (e.g. from the host cell culture medium).
The invention also provides a composition comprising the humanized anti-CD11a antibody and a pharmaceutically acceptable carrier or diluent. This composition for therapeutic use is sterile and may be lyophilized. The invention further provides a method for treating a mammal suffering from a LFA-1 mediated disorder, comprising administering a pharmaceutically effective amount of the humanized anti-CD11a antibody to the mammal. For such therapeutic uses, other immunosuppressive agents or adhesion molecule antagonists (e.g. another LFA-1 antagonist or a VLA-4 antagonist) may be co-administered to the mammal either before, after, or simultaneously with, the humanized anti-CD11a antibody.